The betagamma subunits from heterotrimeric G proteins occupy a central position in the G protein-coupled receptor system. They are multifaceted proteins that have determinants to specify interactions with G protein-coupled receptors, G protein alpha subunits, and multiple downstream target effector molecules. While the three dimensional structure for this protein has been solved, the structural and biochemical features that are responsible for functional interactions with protein partners are not understood. A major goal of our laboratory has been to understand the nature of the interactions between betagamma subunits and its various binding partners. In the course of the last funding period we obtained evidence that G protein betagamma subunits have a unique surface, with properties similar to protein-protein interaction "hot spots," that is critical for mediating effector recognition and regulation. Surprisingly, we found that peptides that bind to this surface activate signal transduction pathways in living cells via a mechanism that does not involve receptors or nucleotide exchange on the alpha subunit. G protein coupled receptor and nucleotide exchange-independent mechanisms for activation of G protein signaling are emerging as playing important regulatory roles in biology. Our identification of a novel mechanism for receptor independent G protein activation suggests another pathway that could be utilized in a cellular context to regulate G protein betagamma subunit signaling. This proposal will explore the mechanisms and physiological implications for this novel G protein activation process in the following specific aims: 1. Determination of the nature of the protein recognition surface on betagammasubunits and its role in a novel nucleotide exchange-independent mechanism for betagamma subunit release. 2. Determination of the mechanism for activation of betagamma-dependent signal transduction in living cells by cell permeable peptides that mediate nucleotide exchange-independent G protein subunit dissociation. 3. Identification of cellular factors that mediate nucleotide exchange-independent subunit dissociation. 4. Identification of sites of interaction G protein betagamma subunits with cellular targets using deuterium exchange mass spectrometry.